Oxidative stress appears to play an important role in degeneration of dopaminergic neurons of the substantia nigra (SN) associated with Parkinson's disease (PD). The SN of early PD patients have dramatically decreased levels of the thiol tripeptide glutathione (GSH). GSH plays multiple roles in the nervous system both as an antioxidant and a redox modulator. Recently, we generated dopaminergic cell lines in which levels of GSH can be inducibly down-regulated via doxycycline (dox) induction of antisense messages against both the heavy and light subunits of gamma glutamyl cysteine synthetase (gamma-GCS), the rate-limiting enzyme in glutathione synthesis. Down-regulation of GCS results in reduction in mitochondrial GSH levels, increased oxidative stress, and decreased mitochondrial function. Interestingly, decreases in mitochondrial activities in GSH-depleted PC12 cells appears to be due to a selective inhibition of complex I activity similar to that observed in PD. This loss in enzymatic activity appears to be a result of cysteine oxidation which is reversible by the thiol-reducing agent dithiothreitol. These results suggest that early observed GSH losses in PD may be directly responsible for the noted decreases in complex I activity and the subsequent mitochondrial dysfunction which ultimately leads to dopaminergic cell death associated with the disease. The hypothesis we will examine in this proposal is that oxidation of specific cysteines within the protein subunits of mitochondrial complex I are responsible for the selective inhibition of its activity following GSH depletion. To accomplish this goal, we will employ a series of sulfhydryl-specific probes to assess the redox states of cysteine thiol groups in complex I proteins. We will use highly sensitive mass spectrometry-based proteomics methods to identify the cysteine residue(s) that are responsible for this reversible loss of mitochondrial complex I activity. We will also examine complex I proteins for other types of oxidative damage (both reversible and irreversible) that may contribute to this loss of activity. These data should provide valuable insight into the effect of oxidative stress on mitochondrial physiology as it relates to PD, particularly the structural basis for alterations in mitochondrial function. Knowledge of the molecular details of complex I dysfunction and the identification specific subunit(s) that are involved may point us towards novel therapeutic targets for the disease and provide key data on whether thiol replacement therapy is a viable option for treatment of the disease. Once identified, presence of these alterations will be assessed in future years in both an antiGSH transgenic mouse model of Parkinson disease as well as in Parkinsonian brains.